Sex-dependent differences in macaque brain mitochondria.

Mitochondrial bioenergetics in females and males is different. However, whether mitochondria from male and female brains display differences in enzymes of oxidative phosphorylation remains unknown. Therefore, we characterized mitochondrial complexes from the brains of male and female macaques (Macaca mulatta). Cerebral tissue from male macaques exhibits elevated content and activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) and higher activity of complex II (succinate dehydrogenase) compared to females. No significant differences between sexes were found in the content of α-ketoglutarate dehydrogenase or in the activities of cytochrome c oxidase and F1Fo ATPase. Our results underscore the need for further investigations to elucidate sex-related mitochondrial differences in humans.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

Body-Wide Inactivation of the Myc-Like Mlx Transcription Factor Network Accelerates Aging and Increases the Lifetime Cancer Incidence.

The "Mlx" and "Myc" transcription factor networks cross-communicate and share many common gene targets. Myc's activity depends upon its heterodimerization with Max, whereas the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. The current work demonstrates that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability, and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a higher cancer incidence. Like Myc, the expression of Mlx, MondoA, and ChREBP and their control over their target genes deteriorate with age in both mice and humans. Collectively, these findings underscore the importance of lifelong and balanced cross-talk between the two networks to maintain proper function and regulation of the many factors that can affect normal aging.

Two enzymes contribute to citrate production in the mitochondrion of Toxoplasma gondii.

Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities and two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses on the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, deletion of CS1 resulted in significantly reduced flux of glucose derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites, or there are other routes of citrate supply in the parasite mitochondrion.

Lactate promotes fatty acid oxidation by the TCA cycle and mitochondrial respiration in muscles of obese mice.

Lower oxidative capacity in skeletal muscles (SKMs) is a prevailing cause of metabolic diseases. Exercise not only enhances the fatty acid oxidation (FAO) capacity of SKMs but also increases lactate levels. Given that lactate may contribute to tricarboxylic acid cycle (TCA) flux and impact monocarboxylate transporter 1 in the SKMs, we hypothesize that lactate can influence glucose and fatty acid (FA) metabolism. To test this hypothesis, we investigated the mechanism underlying lactate-driven FAO regulation in the SKM of mice with diet-induced obesity (DIO). Lactate was administered to DIO mice immediately after exercise over three weeks. We found that increased lactate levels enhanced energy expenditure mediated by fat metabolism during exercise recovery and decreased triglyceride levels in DIO mice SKMs. To determine the lactate-specific effects without exercise, we administered lactate to mice on a high-fat diet (HFD) for eight weeks. Similar to our exercise conditions, lactate increased FAO, TCA cycle activity, and mitochondrial respiration in the SKMs of HFD-fed mice. Additionally, under sufficient FA conditions, lactate increased uncoupling protein-3 abundance via the NADH/NAD+ shuttle. Conversely ATP synthase abundance decreased in the SKMs of HFD mice. Taken together, our results suggest that lactate amplifies the adaptive increase in FAO capacity mediated by the TCA cycle and mitochondrial respiration in SKMs under sufficient FA abundance.

Abscisic acid-mediated guard cell metabolism regulation.

Abscisic acid (ABA) is crucial for plant water deficit (WD) acclimation, but how the interplay between ABA and guard cell (GC) metabolism aids plant WD acclimation remains unclear. Here, we investigated how ABA regulates GC metabolism and how this contributes to plant WD acclimation using tomato wild type (WT) and the ABA-deficient sitiens mutant. These genotypes were characterized at physiological, metabolic, and transcriptional levels under recurring WD periods and were used to perform a13C-glucose labelling experiment using isolated guard cells following exogenously applied ABA. ABA deficiency altered the level of sugars and organic acids in GCs in both irrigated and WD plants and the dynamic of accumulation/degradation of these compounds in GCs during the dark-to-light transition. WD-induced metabolic changes were more pronounced in sitiens than WT GCs. Results from the 13C-labelling experiment indicate that ABA is required for the glycolytic fluxes toward malate and acts as a negative regulator of a putative sucrose substrate cycle. The expression of key ABA-biosynthetic genes was higher in WT than in sitiens GCs after two cycles of WD. Additionally, the intrinsic leaf water use efficiency increased only in WT after the second WD cycle, compared to sitiens. Our results highlight that ABA deficiency disrupts the homeostasis of GC primary metabolism and the WD memory, negatively affecting plant WD acclimation. Our study demonstrates which metabolic pathways are activated by WD and/or regulated by ABA in GCs, which improves our understanding of plant WD acclimation, with clear consequences for plant metabolic engineering in the future.

Is the TCA cycle malate dehydrogenase-citrate synthase metabolon an illusion?

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques.

Replenishment of TCA cycle intermediates and long-noncoding RNAs regulation in breast cancer.

The tricarboxylic acid (TCA) cycle is an essential interface that coordinates cellular metabolism and is as a primary route determining the fate of a variety of fuel sources, including glucose, fatty acid and glutamate. The crosstalk of nutrients replenished TCA cycle regulates breast cancer (BC) progression by changing substrate levels-induced epigenetic alterations, especially the methylation, acetylation, succinylation and lactylation. Long non-coding RNAs (lncRNA) have dual roles in inhibiting or promoting energy reprogramming, and so altering the metabolic flux of fuel sources to the TCA cycle, which may regulate epigenetic modifications at the cellular level of BC. This narrative review discussed the central role of the TCA cycle in interconnecting numerous fuels and the induced epigenetic modifications, and the underlying regulatory mechanisms of lncRNAs in BC.

Hypoxia makes EZH2 inhibitor not easy-advances of crosstalk between HIF and EZH2.

Histone methylation plays a crucial role in tumorigenesis. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that regulates chromatin structure and gene expression. EZH2 inhibitors (EZH2is) have been shown to be effective in treating hematologic malignancies, while their effectiveness in solid tumors remains limited. One of the major challenges in the treatment of solid tumors is their hypoxic tumor microenvironment. Hypoxia-inducible factor 1-alpha (HIF-1α) is a key hypoxia responder that interacts with EZH2 to promote tumor progression. Here we discuss the implications of the relationship between EZH2 and hypoxia for expanding the application of EZH2is in solid tumors.

Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i.

BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.

Associations between TCA cycle plasma metabolites and fatigue in black females with systemic lupus erythematosus: An untargeted metabolomics pilot study.

OBJECTIVE: In this pilot study, we used untargeted metabolomics to identify biochemical mechanisms or biomarkers potentially underlying SLE-related fatigue. METHODS: Metabolon conducted untargeted metabolomic plasma profiling using ultrahigh performance liquid chromatography/tandem mass spectrometry on plasma samples of 23 Black females with systemic lupus erythematosus (SLE) and 21 no SLE controls. Fatigue phenotypes of general fatigue, physical fatigue, mental fatigue, reduced activity, and reduced motivation were measured with the reliable and valid Multidimensional Fatigue Inventory (MFI). RESULTS: A total of 290 metabolites were significantly different between the SLE and no SLE groups, encompassing metabolites related to glycolysis, TCA cycle activity, heme catabolism, branched chain amino acids, fatty acid metabolism, and steroids. Within the SLE group, controlling for age and co-morbidities, TCA cycle metabolites of alpha-ketoglutarate (AKG) and succinate were statistically significantly associated (p < .05) with physical and general fatigue. CONCLUSION: While pervasive perturbations in the entire TCA cycle have been implicated as a potential mechanism for fatigue, our results suggest individual metabolites of AKG and succinate may be potential biomarkers or targets of intervention for fatigue symptom management in SLE. Additionally, perturbations in heme metabolism in the SLE group provide additional insights into mechanisms that promote systemic inflammation.

The association of circulating bioenergetic metabolites with healthy human aging.

Aging is an inevitable and gradual decline in several biological functions. Mitochondrial dysfunction is one of the most important hallmarks of aging. In this context, alterations in metabolites associated with mitochondrial dysfunction may serve as a significant biomarker. This study aimed to investigate the existence of a relationship between the key metabolites involved in bioenergetics metabolism and aging. 53 volunteers ranged 20-85 years participated in the study. We tested the association between different tricarboxylic acid (TCA) cycle metabolites, fatty acid metabolism, and amino acid metabolism with age, sex, body composition, and proxy markers of aging such as walking speed, grip strength and chair test. We found that lactic acid negatively correlated with age while several fatty acid metabolites, such as azelaic, sebacic, and linoleic acids, showed positive correlations with age (p < 0.05). Sex-specific trends, such as glycerol, and dodecanoic acid, were also observed for certain metabolites. Furthermore, citric acid levels were found to have a significant association with physical function and body composition measures. Participants with higher citric acid levels displayed improved performance in physical tests and favorable body composition indices. Additionally, fumaric acid and adipic acid showed positive correlations with fat-free body mass, while sebacic acid was negatively associated with measures of fat mass. These findings underscore the importance of understanding the role of circulating bioenergetics metabolites with age, sex variations, and their potential implications in body composition and physical performance.

Metabolic adaptation in epithelial ovarian cancer metastasis.

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.

Ovarian aging: energy metabolism of oocytes.

In women who are getting older, the quantity and quality of their follicles or oocytes and decline. This is characterized by decreased ovarian reserve function (DOR), fewer remaining oocytes, and lower quality oocytes. As more women choose to delay childbirth, the decline in fertility associated with age has become a significant concern for modern women. The decline in oocyte quality is a key indicator of ovarian aging. Many studies suggest that age-related changes in oocyte energy metabolism may impact oocyte quality. Changes in oocyte energy metabolism affect adenosine 5'-triphosphate (ATP) production, but how related products and proteins influence oocyte quality remains largely unknown. This review focuses on oocyte metabolism in age-related ovarian aging and its potential impact on oocyte quality, as well as therapeutic strategies that may partially influence oocyte metabolism. This research aims to enhance our understanding of age-related changes in oocyte energy metabolism, and the identification of biomarkers and treatment methods.

Reprogrammed mitochondria: a central hub of cancer cell metabolism.

Mitochondria represent the metabolic hub of normal cells and play this role also in cancer but with different functional purposes. While cells in differentiated tissues have the prerogative of maintaining basal metabolism and support the biosynthesis of specialized products, cancer cells have to rewire the metabolic constraints imposed by the differentiation process. They need to balance the bioenergetic supply with the anabolic requirements that entail the intense proliferation rate, including nucleotide and membrane lipid biosynthesis. For this aim, mitochondrial metabolism is reprogrammed following the activation of specific oncogenic pathways or due to specific mutations of mitochondrial proteins. The main process leading to mitochondrial metabolic rewiring is the alteration of the tricarboxylic acid cycle favoring the appropriate orchestration of anaplerotic and cataplerotic reactions. According to the tumor type or the microenvironmental conditions, mitochondria may decouple glucose catabolism from mitochondrial oxidation in favor of glutaminolysis or disable oxidative phosphorylation for avoiding harmful production of free radicals. These and other metabolic settings can be also determined by the neo-production of oncometabolites that are not specific for the tissue of origin or the accumulation of metabolic intermediates able to boost pro-proliferative metabolism also impacting epigenetic/transcriptional programs. The full characterization of tumor-specific mitochondrial signatures may provide the identification of new biomarkers and therapeutic opportunities based on metabolic approaches.

Mechanisms associated with cuproptosis and implications for ovarian cancer.

Ovarian cancer, a profoundly fatal gynecologic neoplasm, exerts a substantial economic strain on nations globally. The formidable challenge of its frequent relapse necessitates the exploration of novel cytotoxic agents, efficacious antineoplastic medications with minimal adverse effects, and strategies to surmount resistance to primary chemotherapeutic agents. These endeavors aim to supplement extant pharmacological interventions and elucidate molecular mechanisms underlying induced cytotoxicity, distinct from conventional therapeutic modalities. Recent scientific research has unveiled a novel form of cellular demise, known as copper-death, which is contingent upon the intracellular concentration of copper. Diverging from conventional mechanisms of cellular demise, copper-death exhibits a pronounced reliance on mitochondrial respiration, particularly the tricarboxylic acid (TCA) cycle. Tumor cells manifest distinctive metabolic profiles and elevated copper levels in comparison to their normal counterparts. The advent of copper-death presents alluring possibilities for targeted therapeutic interventions within the realm of cancer treatment. Hence, the primary objective of this review is to present an overview of the proteins and intricate mechanisms associated with copper-induced cell death, while providing a comprehensive summary of the knowledge acquired regarding potential therapeutic approaches for ovarian cancer. These findings will serve as valuable references to facilitate the advancement of customized therapeutic interventions for ovarian cancer.

The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Elevated PINK1/Parkin-Dependent Mitophagy and Boosted Mitochondrial Function Mediate Protection of HepG2 Cells from Excess Palmitic Acid by Hesperetin.

Deregulation of mitochondrial functions in hepatocytes contributes to many liver diseases, such as nonalcoholic fatty liver disease (NAFLD). Lately, it was referred to as MAFLD (metabolism-associated fatty liver disease). Hesperetin (Hst), a bioactive flavonoid constituent of citrus fruit, has been proven to attenuate NAFLD. However, a potential connection between its preventive activities and the modulation of mitochondrial functions remains unclear. Here, our results showed that Hst alleviates palmitic acid (PA)-triggered NLRP3 inflammasome activation and cell death by inhibition of mitochondrial impairment in HepG2 cells. Hst reinstates fatty acid oxidation (FAO) rates measured by seahorse extracellular flux analyzer and intracellular acetyl-CoA levels as well as intracellular tricarboxylic acid cycle metabolites levels including NADH and FADH2 reduced by PA exposure. In addition, Hst protects HepG2 cells against PA-induced abnormal energetic profile, ATP generation reduction, overproduction of mitochondrial reactive oxygen species, and collapsed mitochondrial membrane potential. Furthermore, Hst improves the protein expression involved in PINK1/Parkin-mediated mitophagy. Our results demonstrate that it restores PA-impaired mitochondrial function and sustains cellular homeostasis due to the elevation of PINK1/Parkin-mediated mitophagy and the subsequent disposal of dysfunctional mitochondria. These results provide therapeutic potential for Hst utilization as an effective intervention against fatty liver disease.

Black raspberry-mediated metabolic changes in patients with familial adenomatous polyposis associated with rectal polyp regression.

Familial adenomatous polyposis (FAP) patients face an almost certain 100% risk of developing colorectal cancer, necessitating prophylactic colectomy to prevent disease progression. A crucial goal is to hinder this progression. In a recent clinical trial involving 14 FAP patients, half received 60 g of black raspberry (BRB) powder orally and BRB suppositories at bedtime, while the other half received only BRB suppositories at bedtime over 9 months. This intervention led to a notable reduction in rectal polyps for 11 patients, although 3 showed no response. In this study, we delved into the metabolic changes induced by BRBs in the same patient cohort. Employing mass spectrometry-based non-targeted metabolomics, we analyzed pre- and post-BRB urinary and plasma samples from the 11 responders. The results showed significant alterations in 23 urinary and 6 plasma metabolites, influencing various pathways including polyamine, glutathione metabolism, the tricarboxylic acid cycle, inositol metabolism, and benzoate production. BRBs notably elevated levels of several metabolites associated with these pathways, suggesting a potential mechanism through which BRBs facilitate rectal polyp regression in FAP patients by modulating multiple metabolic pathways. Notably, metabolites derived from BRB polyphenols were significantly increased post-BRB intervention, emphasizing the potential therapeutic value of BRBs in FAP management.

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.